WO1999051217A1 - Inhibition of binding of hox and homeodomain-containing proteins and uses thereof - Google Patents

Inhibition of binding of hox and homeodomain-containing proteins and uses thereof Download PDF

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Publication number
WO1999051217A1
WO1999051217A1 PCT/US1999/007455 US9907455W WO9951217A1 WO 1999051217 A1 WO1999051217 A1 WO 1999051217A1 US 9907455 W US9907455 W US 9907455W WO 9951217 A1 WO9951217 A1 WO 9951217A1
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smadl
hoxc
binding
gene
interaction
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PCT/US1999/007455
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English (en)
French (fr)
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Xu Cao
Xingming Shi
Zhijie Chang
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The Uab Research Foundation
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Priority to JP2000541988A priority Critical patent/JP2002510620A/ja
Priority to AU34708/99A priority patent/AU757105B2/en
Priority to NZ507251A priority patent/NZ507251A/en
Priority to EP99916373A priority patent/EP1075255A4/en
Priority to CA002324976A priority patent/CA2324976A1/en
Priority to IL13883299A priority patent/IL138832A0/xx
Publication of WO1999051217A1 publication Critical patent/WO1999051217A1/en
Priority to NO20005022A priority patent/NO20005022L/no

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

  • the present invention relates generally to the fields of transcriptional regulation. More specifically, the present invention relates to inhibition of binding of hox and homeodomain- containing proteins and uses thereof.
  • the bone morphogenetic proteins (BMPs), a subfamily of TGF ⁇ , are potent growth factors that regulate embryonic development, vertebral patterning and mesenchymal cell differentiation (10,11).
  • BMP-2/4 identified as bone inductive growth factors, are important signaling molecules during development of the skeleton in vertebrates ( 12-14).
  • Smad l protein Central to the bone morphogenetic protein signaling pathway is the Smad l protein, which translocates into the nucleus to regulate gene transcription upon direct phosphorylation by bone morphogenetic protein receptors (1 ,3,4).
  • TGF ⁇ superfamily Growth factors in the TGF ⁇ superfamily have b een implicated in various processes during vertebrate embryonic development.
  • the TGF ⁇ action on induction and patterning of mesoderm and skeletal development has been studied intensely.
  • the TGF ⁇ related molecules, BMP-2/4 induces skeletal patterning, growth of limb buds and skeletal cell differentiation.
  • Hox and homeodomain containing transcription factors are also involved in the same process, and have been suggested as a downstream regulation of BMP-4 to mediate its effects.
  • hox proteins are DNA binding proteins, very little is known about their natural DNA response elements and their role in transcription.
  • Northern blot analysis shows that Hoxc-8 is expressed during human embryo development a t high levels in spinal cord, backbone and limbs and at a lower level in heart (20).
  • BMP-2/4 activates expression of Hox genes, induces osteoblast differentiation and controls patterning across th e anteroposterior (a-p) axis of developing limb (21 ).
  • the prior art is deficient in methods for stimulating osteoblast differentiation and bone formation.
  • the prior art is also deficient in methods of regulating transcription via the Hox proteins and/or homeobox-containing proteins.
  • the pre sent invention fulfills this long-standing need and desire in the art.
  • the bone morphogenetic protein-2 (BMP-2) w as identified as a bone inductive growth factor, involved in inducing osteoblast differentiation.
  • BMP-2 The bone morphogenetic protein-2
  • the present invention identifies a specific interaction of Smadl with Hox and homeodomain-containing proteins, which often act as transcriptional repressors, in which the binding of Smadl to Hoxc-8 inhibits the recognition an d binding of Hoxc-8 to its DNA binding site in a dose-dependent manner.
  • This specific interaction between Smadl and Hoxc-8 can be used as a target to inhibit the binding of Hoxc-8 to its DNA binding sites, thereby inducing osteoblast differentiation an d preventing osteoporosis.
  • the interaction of Smadl with other Hox proteins or homeobox-containing proteins may also be used to regulate other diseases, such as cancer or cardiovascular disease.
  • a method of stimulating bone formation in an individual comprising the step of: inducing an interaction between Smad l and a homeobox-containing transcription factor.
  • this interaction induces a BMP-responsive gene which encodes a bone matrix protein. This induction results in osteoblast and/ or chondroblast differentiation, which in turn, stimulates bone formation.
  • a method of inducing gene(s) encoding bone matrix proteins comprising the step of: inducing an interaction between Smadl and a homeobox-containing transcription factor in which the interaction results in an induction of gene(s) encoding bone matrix proteins.
  • a method of inducing a gene encoding osteopontin comprising the steps of: inducing an interaction between Smadl and Hoxc-8. Preferably, this interaction results in removal of the transcriptional repression and induction of the gene encoding osteopontin.
  • a method of screening for a compound that stimulates bone formation comprising the steps of: contacting a cell with a compound; and determining the ability of the compound to inhibit binding of Hoxc-8 to a gene. This inhibition of binding results in induction of the gene which indicates that th e compound stimulates bone formation.
  • a method of regulating disease in an individual comprising the step of: inhibiting the binding of a homeobox- containing transcription factor to a gene involved in regulating disease, wherein the inhibition removes transcriptional repression of the gene by the homeobox-containing protein and results in th e induction of those genes involved in regulating disease.
  • Figure 1 shows the specific interactions of Smad l with Hoxc-8 in a two-hybrid system.
  • Figure 1A shows the two- hybrid growth assay of interaction between Smadl and Hoxc-8. Specific interactions were noted in yeast bearing both pGBT9- Smadl and PAC-Hoxc-8 plasmids, which grew on media lacking His, Leu and Trp.
  • Figure IB shows the ⁇ -gal liquid assay for two- hybrid assays. ⁇ -galactosidase activities for yeast bearing plasmids as indicated were plotted.
  • Figure 1C shows the specific interaction of Smadl with Hoxc-8 in a pulldown experiment.
  • Hoxc-8 protein was labeled with [ 35 S] methionine by translation and incubated with purified GST-Smadl or GST-protein. Samples were subsequently incubated with GST-Sepharose, washed, eluted in SDS buffer and separated on 10% SDS-PAGE gels.
  • Figure 2 shows the interaction of Smadl with Hoxc-8 inhibits Hoxc-8 function as a repressor.
  • Figure 2A shows that Hoxc-8 specifically binds to the Hox DNA binding element.
  • EMSA was performed using 32 P-labeled Hox binding element alone (lane 1) or with GST (lane 2) and GST-Hoxc-8 (lanes 3-10).
  • Lanes 4 - 6 and 8-10 contained 5-, 25-, and 100-fold molar excess of unlabeled Hox element (Probe-S) and MSX-2 DNA binding element
  • FIG. 6 shows that Smadl inhibits binding of Hoxc- 8 to its DNA binding element in a dose-dependent manner.
  • EMSA was performed using 32 P-labeled Hox binding element alone (lane 1), with GST (lane 2), GST-Smadl(lane 3) or GST-Hoxc-8 protein (lanes 4-8) and different amounts of GST-Smadl (lanes 5-8) .
  • Figure 2C shows the interaction of both Smadl and 3 with Hoxc- 8 in vivo. FLAG-tagged Smadl and -4 and HA-tagged Hosc-8 w ere co-transfected with or without ALK3 (Q233D).
  • Figure 3 shows the characterization of a Hoxc-8 DNA binding site from osteopontin promoter.
  • Figure 3 A shows th e DNA probes of osteopontin promoter that were used for EMSA in Figure 3B , 3C, 3D, 3E, 3F and 3G.
  • Figure 3B shows that th e OPN-2 DNA fragment contains a Hoxc-8 binding site.
  • EMSA w as performed using different 32 P-labeled DNA fragments: OPN-1 (lanes 1-3), OPN-2 (lanes 4-6) and OPN-3 (lanes 7-9), an d incubated with probe alone (lanes 1 , 4 and 7), GST (lanes 2, 5, and 8) or GST-Hoxc-8 (lanes 3, 6 and 9).
  • FIG. 3C shows that OPN-5 is the Hoxc-8 binding site.
  • EMSA was performed using smaller 32 P-labeled DNA fragments: OPN-4 (lanes 1-5), OPN-5 (lanes 6- 10) and OPN-6 (lanes 11-15), and incubated with probe alone (lanes 1 , 6 and 11), GST (lanes 2, 7, and 12), GST-Smadl (lanes 3, 5, 8, 10, 13 and 15) or GST-Hoxc-8 (lanes 4,5, 9, 10, 14, and 15).
  • Figure 3D shows that Hoxc-8 specifically binds to OPN-5.
  • EMSA w as
  • Lanes 3-5 and 6-8 contained 5-, 25-, and 100-fold molar excess of unlabeled OPN-5 and MSX-2 DNA binding element (Probe-M).
  • Figure 3E shows that Smadl inhibits binding of Hoxc-8 to OPN-5 in a concentration-dependent manner. EMSA was performed using 3 P-labeled OPN-5 alone (lane 1), with 1.5 ⁇ g GST (lane 2), 1.5 ⁇ g GST-Smadl (lane 3) or 0.2 ⁇ g GST-Hoxc-8 protein (lanes 4-7) and different amounts of GST-Smadl (1.5, 3 and 4.5 ⁇ g for lanes 5-7, respectively).
  • FIG. 3F shows that Hox proteins interact with Smadl and -4 but ot Smad2 and -3.
  • Hoxa-9 and Hosc-8 GST fusion proteins (0.2 ⁇ g) were tested for their ability to interact with Smadl , -2, -3 and -4 or GST (3 ⁇ g) in a gel shift assay.
  • Figure 3G shows that Smads do not inhibit binding of Msx-1 and Msx-2 homeodomains containing proteins to their cognate DNA element.
  • Purified GST-Msx-1 or -Msx-2 (0.5 ⁇ g) w as incubated together with probe-M and different Smads (3 ⁇ g) .
  • Figure 4 shows that BMP-2-induced osteopotin gene transcription is mediated by a Hoxc-8 binding site.
  • Figure 4 A shows a schematic description of the constructs used in the transfection assays: OPN-266 is the native osteopontin construct; Hox-pGL3 contains the osteopontin Hox binding site linked to th e SV40 promoter; mHOX-pGL3 contains the mutated osteopontin Hox binding site.
  • Figure 4B shows that BMP activates the osteopontin promoter.
  • the OPN-266 plasmid was co-transfected in C3H10T1/2 mesenchymal cells with Hoxc-8, Smadl, or Smad4 plasmids alone or in a combination of all three in the presence of absence of ALK3 plasmid.
  • Figure 4C shows the osteopontin Hox binding site mediates BMP-induced transcription.
  • Hox-pGL3 construct was co-transfected with ALK6 or ALK3 in C3H 10T 1/2 mesenchymal cells.
  • Figure 4D shows that mutation of Hox binding site abolishes BMP stimulation.
  • Hox-pGL3 construct or mHox-pGL3-pGL3 control plasmid was co-transfected with ALK6, ALK3 or Hoxc-8 plasmids in C3H10T1/2 mesenchymal cells.
  • Cell lysates in panels B, C, and D were assayed for luciferase activity normalized to Renilla luciferase levels 48 h after transfection. Experiments were repeated twice in triplicate.
  • Figure 5 shows that the N-terminal domains of
  • FIG. 5 A shows an SDS-PAGE profile of purified GST-Smadl fragments used in the gel shift assays shown in panel B and schematic presentation of Smad l deletion constructs.
  • Bacterial expressed GST recombinant Smad l proteins as indicated in amino acid numbers were purified on glutathione-agarose. Glutathione elutions were loaded onto a 10% SDS-PAGE and visualized by Coomassie Blue staining (left panel) . The sizes of each were verified with the molecular mass markers on the top lane of the gel.
  • Figure 5B shows that two regions of Smadl confer the inhibitory effect on Hoxc-8 binding.
  • Hoxc-8 was incubated with the same probe in the absence (lane 1) and the presence of Smadl fragments 1 0 1 - 145 (lanes 2-4) or 148-191 (lanes 5-7) with a 2-fold increase in truncated Smadl concentration between successive lanes.
  • Figure 6 shows that the homeodomain of Hoxc-8 interacts with Smadl .
  • Figure 6A shows schematic illustrations of various deletion mutants of Hoxc-8 used for interaction studies . The size of each is labeled by the amino acid residues. CR1, conserved region 1 ; HP, hexapeptide; HDC, homeodomain and its C- terminal extension; and HD, homeodomain.
  • FIG. 6B shows th e interaction between Smadl and Hoxc-8 in yeast two-hybrid system.
  • Yeast strain Y190 containing the plasmid pGBT9/Smadl was transformed with pACT2 (control) or pACT2 containing various-sized Hoxc-8 cDNA as indicated.
  • Transformants colony were assayed for the ⁇ -galactosidase activities and the values were normalized for the cell densities. Each bar represents th e mean ( SD from three independent determinations.
  • Figure 6C shows that the homeodomain and its C-terminal extension of Hoxc-8 are involved in the interaction with Smadl MHl domain.
  • Figure 7 shows the Smadl domains containing Hoxc-8 interaction regions induce bone cell differentiation.
  • Figure 7 A shows the constructs of Smadl-NL, Smadl-L, and Smad l -M.
  • pTet-Splice vector was used to make tetracyclin (Tet)-regulated mammalian expression plasmids for Smadl-NL (aa 3-276), -L (aa 145-276), and -M (aa 104- 191).
  • a nuclear localization signal (NLS) was fused to each construct allow the expressed truncated proteins to enter the nucleus.
  • Figure 7B shows that expression of Smadl fragments is Tet regulated.
  • RNA from panel A was extracted after 2-day culturing the cells in the absence or presence of Tet.
  • the expression of each Smad l fragment was induced upon Tet withdrawal.
  • Figure 7C shows that alkaline phosphatase activity is induced by the Hoxc-8 interaction domains of Smadl .
  • 2T3 cells bearing pTet-Splice vector (vector), pTet-Splice/Smadl-NL (Smadl-NL), -Smadl-L, or Smadl-M was cultured in the presence or absence of Tet and the cells were lysed at indicated days (x-axis). Alkaline phosphatase activity was determined as described herein and the values w ere normalized for the protein contents. Each bar represents at least 3 independent measurements.
  • Figure 7D shows that Smad l fragments induce mineral matrix formation in 2T3 clones. Indicated stable clones were cultured in the presence or absence of Tet or in the presence of 100 ng/ml BMP-2 (BMP-2) for 1 2 days. Cells then were fixed and stained by the von Kossa method. Mineral crystals (black spots) were formed cells treated with
  • FIG 8 shows that overexression of Hoxc-8 induces a high level of alkaline phosphatase activity.
  • 2T3 cells w ere stably transfected with a mammalian expression plasmid for Hoxc-8 (pcDNA3/Hoxc-8). Positive clones were selected by the Slot-Blot hybridization and assayed for their alkaline phosphatase activity as described in Figure 7. Means and SDs of triplicates are shown.
  • Figure 9 shows a proposed model for the mechanism of Hoxc-8 interaction domains of Smadl mimicking BMP signaling and inducing bone cell differentiation. Hoxc-8 represses gene transcription in the basal state in the pluripotent and self- renewable stem cells.
  • Smadl-Hoxc-8 interaction domains When the Smadl-Hoxc-8 interaction domains are expressed, they enter the nucleus and bind to Hoxc-8.
  • the interaction of Smadl fragments with Hoxc-8 inhibits Hoxc-8 binding to its cognate DNA element in the bone marker genes (such as osteopontin) which then derepresses Hoxc-8 and activates gene transcription and subsequently induces the formation of osteoblast cells.
  • bone marker genes such as osteopontin
  • TGF- ⁇ superfamily requires the interaction of two types of serine/threonine transmembrane kinase receptors (1).
  • the signaling is mediated by direct phosphorylation of Smad proteins. Phosphorylation of Smad2 and
  • Smad3 is by TGF ⁇ and activin (2,3), whereas Smadl and Smad5
  • bone morphogenetic proteins which are members of the TGF- ⁇ superfamily (4,5).
  • Smad proteins Upon phosphorylation, the Smad proteins interact with a common partner, Smad4, and translocate into the nucleus where the complex recruits DNA binding protein(s) to activate specific gene transcription (4,6-9).
  • Smad4 a common partner
  • DNA binding protein(s) involved in bone morphogenetic protein signaling have not been identified.
  • This invention demonstrates that Smadl specifically interacts with Hoxc-8, a member of the homeodomain transcription factor family, inhibiting binding of Hoxc-8 to its DNA binding site in a dose dependent manner.
  • Hoxc-8 functions as a transcriptional repressor and is predominantly expressed in bone tissues.
  • a Hoxc-8 binding site has been characterized from the 5'-flanking region of the osteopontin gene, whose expression is rapidly induced by BMP-2/4. It appears that bone morphogenetic protein-induced osteopontin gene transcription is mediated through the Hoxc-8 binding site.
  • the present invention is directed towards a method of stimulating bone formation in an individual, comprising the step of: inducing an interaction between Smadl and a homeobox- containing transcription factor, wherein the interaction induces a BMP-responsive gene encoding a bone matrix protein which results in osteoblast and/or chondroblast differentiation, which subsequently stimulates bone formation.
  • Representative means of inducing the interaction include phosphorylation of Smad l , overexpression of Smadl , and mutation of the homeobox- containing transcription factor.
  • the present invention is also directed towards a method of inducing gene(s) encoding bone matrix proteins, comprising the step of: inducing an interaction between Smad l and a homeobox-containing transcription factor in which the interaction results in an induction of gene(s) encoding bone matrix proteins. Representative means of induction are described above, as are representative homeobox-containing transcription factors and BMP-responsive genes.
  • the present invention is directed towards a method of inducing a gene encoding osteopontin, comprising the steps of: inducing an interaction between Smadl and Hoxc-8, wherein the interaction results in removing transcriptional repression of a gene encoding osteopontin which induces the gene encoding osteopontin.
  • the present invention is still further directed towards a method of screening for a compound that stimulates bone formation, comprising the steps of: contacting a cell with a compound; and determining the ability of the compound to inhibit binding of Hoxc-8 to a gene. Inhibition of binding results in induction of the gene, which is indicative of a compound that stimulates bone formation.
  • Representative compounds include a n antibody or fragment thereof, synthetic drugs, synthetic proteins or a phosphorylated form of Smadl or fragments thereof. Inhibition of binding can be determined by methods such as a gel- shift assay, transcription, Northern blotting, and Western blotting.
  • the present invention is additionally directed towards a method of regulating disease in an individual, comprising the step of: inhibiting the binding of a homeobox-containing transcription factor to a gene involved in regulating disease in cells of the individual. Inhibition of binding removes transcriptional repression by the homeobox-containing protein of the gene, thereby resulting in the induction of the genes involved in regulating disease. In this case, inhibition may be due to th e presence of a compound that binds to the homeobox-containing transcription factor, thereby inhibiting the DNA binding ability of the homeobox-containing transcription factor.
  • representative compounds include an antibody or fragment thereof, synthetic drugs, synthetic proteins and a phosphorylated form of Smadl or fragments thereof.
  • Preferred homeobox- containing transcription factor are Hoxc-8, Hoxa-9, Msxl , and Msx2. This method may be applied to individuals with osteoporosis, cancer, cardiovascular disease and neurological disease.
  • BMP-induced gene activation shall refer to any genes that are induced to express upon the stimulation by BMPs.
  • Smadl shall refer to any proteins that are homologous to Drosophila mothers against DPP or MAD protein.
  • interaction between Smadl and Hox or “interaction b etween Smadl and a homeodomain-containing protein” shall refer to any interaction between the two proteins that results in a disruption of the transcription repressor activity of the Hox or homeodomain-
  • transcriptional repression by a hox protein or “transcriptional repression by a homeodomain-containing protein shall refer to any gene whose transcription activities are repressed in the presence of the hox protein or the homeodomain-containing protein.
  • a "DNA molecule” refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its either single stranded form, or a double-stranded helix.
  • This term refers only to the primary and secondary structure of th e molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes.
  • linear DNA molecules e.g., restriction fragments
  • viruses e.g., viruses, plasmids, and chromosomes.
  • a “vector” is a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.
  • a “replicon” is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo; i.e., capable of replication under its own control.
  • An “origin of replication” refers to those DNA sequences that participate in DNA synthesis.
  • An “expression control sequence” is a DNA sequence that controls and regulates the transcription and translation of another DNA sequence.
  • a coding sequence is "operably linked" and “under the control” of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence.
  • expression vectors containing promoter sequences which facilitate the efficient transcription and translation of the inserted DNA fragment are used in connection with the host.
  • the expression vector typically contains an origin of replication, promoter(s), terminator(s), as well as specific genes which are capable of providing phenotypic selection in transformed cells.
  • the transformed hosts can be fermented and cultured according to means known in the art to achieve optimal cell growth.
  • a DNA "coding sequence” is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory
  • a coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences.
  • a polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.
  • a "cDNA” is defined as copy-DNA or complementary-DNA, and is a product of a reverse transcription reaction from an mRNA transcript.
  • An "exon” is an expressed sequence transcribed from the gene locus, whereas an "intron” is a non-expressed sequence that is from th e gene locus.
  • Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers , repressors, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell.
  • a "cis-element” or “DNA binding recognition sequence” is a nucleotide sequence, also termed a “consensus sequence” or “motif, that interacts with other proteins which can upregulate or downregulate expression of a specicif gene locus.
  • a “signal sequence” can also be included with the coding sequence. This sequence encodes a signal peptide, N-terminal to the polypeptide, that communicates to the host cell and directs the polypeptide to the appropriate cellular location. Signal sequences can be found associated with a variety of proteins native to prokaryotes an d eukaryotes .
  • a "promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence.
  • the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription a t levels detectable above background.
  • Within the promoter sequence will be found a transcription initiation site, as well a s protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eukaryotic promoters often, bu t not always, contain "TATA" boxes and "CAT” boxes.
  • Prokaryotic promoters contain Shine-Dalgarno sequences in addition to the - 1 0 and -35 consensus sequences.
  • oligonucleotide is defined as a molecule comprised of two or more deoxyribonucleotides, preferably more than three. Its exact size will depend upon many factors which, in turn, depend upon the ultimate function and use of th e oligonucleotide.
  • primer refers to a n oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand, is induced, i.e., in th e presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH.
  • the primer may be either single-stranded or double-stranded and must b e sufficiently long to prime the synthesis of the desired extension
  • the oligonucleotide primer typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.
  • the primers herein are selected to be “substantially" complementary to different strands of a particular target DNA sequence. This means that the primers must be sufficiently complementary to hybridize with their respective strands . Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5' end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence or hybridize therewith and thereby form the template for the synthesis of the extension product. As used herein, the terms “restriction endonucleases” and “restriction enzymes” refer to enzymes which cut double- stranded DNA at or near a specific nucleotide sequence.
  • Recombinant DNA technology refers to techniques for uniting two heterologous DNA molecules, usually as a result of in vitro ligation of DNAs from different organisms. Recombinant DNA molecules are commonly produced by experiments in genetic engineering. Synonymous terms include “gene splicing",
  • a cell has been "transformed” or “transfected” with exogenous or heterologous DNA when such DNA has been introduced inside the cell.
  • the transforming DNA may or may not be integrated (covalently linked) into the genome of the cell.
  • I n prokaryotes, yeast, and mammalian cells for example, th e transforming DNA may be maintained on an episomal element such as a vector or plasmid.
  • a stably transformed cell is one in which the transforming DNA h as become integrated into a chromosome so that it is inherited b y daughter cells through chromosome replication.
  • a "clone” is a population of cells derived from a single cell or ancestor by mitosis.
  • a “cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations.
  • An organism, such as a plant or animal, th at has been transformed with exogenous DNA is termed "transgenic".
  • the term "host” is meant to include not only prokaryotes but also eukaryotes such as yeast, plant and animal cells.
  • a recombinant DNA molecule or gene can be used to transform a host using any of the techniques commonly known to those of ordinary skill in the art.
  • One preferred embodiment is the use of a vectors containing coding sequences for a gene for purposes of prokaryotic transformation.
  • Prokaryotic hosts m ay include E. coli, S. tymphimurium, Serratia marcescens and Bacillus
  • Eukaryotic hosts include yeasts such as Pichia pastoris, mammalian cells and insect cells, and more preferentially, plant cells, such as Arabidopsis thaliana and Tobaccum nicotiana.
  • Two DNA sequences are "substantially homologous" when at least about 75% (preferably at least about 80%, and most preferably at least about 90% or 95%) of the nucleotides match over the defined length of the DNA sequences. Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II, supra; Nucleic Acid Hybridization, supra.
  • heterologous' region of the DNA construct is a n identifiable segment of DNA within a larger DNA molecule that is not found in association with the larger molecule in nature.
  • th e gene when the heterologous region encodes a mammalian gene, th e gene will usually be flanked by DNA that does not flank th e mammalian genomic DNA in the genome of the source organism.
  • the coding sequence is a construct where th e coding sequence itself is not found in nature (e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene). Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein.
  • fragment as applied to a polypeptide or an antibody, will ordinarily be at least 10 residues, more
  • fragment 22 typically at least 20 residues, and preferably at least 30 (e.g., 50) residues in length, but less than the entire, intact sequence.
  • Fragments can be generated by methods known to those skilled in the art, e.g., by enzymatic digestion of naturally occurring or recombination, by recombinant DNA techniques using a n expression vector that encodes a defined fragment, or by chemical synthesis. The ability of a candidate fragment to exhibit a characteristic of Smadl (e.g., binding to Hoxc-8) can be assessed by methods described herein. Purified fragments of Smadl or antigenic fragments of Smadl can be used to generate antibodies by employing standard protocols known to those skilled in the art.
  • a standard Northern blot assay can be used to ascertain the relative amounts of mRNA in a cell or tissue obtained from plant or other transgenic tissue, in accordance with conventional Northern hybridization techniques known to those persons of ordinary skill in the art.
  • a standard Southern blot assay may be used to confirm the presence and th e copy number of a gene in transgenic systems, in accordance with conventional Southern hybridization techniques known to those of ordinary skill in the art.
  • Both the Northern blot and Southern blot use a hybridization probe, e.g.
  • radiolabelled cDNA either containing the full-length, single stranded DNA or a fragment of that DNA sequence at least 20 (preferably at least 30, more preferably at least 50, and most preferably at least 1 00 consecutive nucleotides in length).
  • the DNA hybridization probe can be labelled by any of the many different methods known to those skilled in this art.
  • the labels most commonly employed for these studies are radioactive elements, enzymes, chemicals which fluoresce when exposed to untraviolet light, and others.
  • a number of fluorescent materials are known and can be utilized as labels. These include, for example, fluorescein, rhodamine, auramine, Texas Red, AMCA blue and Lucifer Yellow.
  • a particular detecting material is anti-rabbit antibody prepared in goats and conjugated with fluorescein through an isothiocyanate. Proteins can also b e labeled with a radioactive element or with an enzyme. The radioactive label can be detected by any of the currently available counting procedures.
  • the preferred isotope may be selected from 3H, 14C, 32p, 35 S , 36 ⁇ , 51 Cr , 57 Co , 58 Co , 59 Fe , 90 ⁇ ? 125 13 1 ⁇ , and 186Re .
  • Enzyme labels are likewise useful, and can be detected by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques .
  • the enzyme is conjugated to the selected particle by reaction with bridging molecules such as carbodiimides, diisocyanates, glutaraldehyde and the like. Many enzymes which can be used in these procedures are known and can be utilized. The preferred are peroxidase, ⁇ -glucuronidase, ⁇ -D-glucosidase, ⁇ -D- galactosidase, urease, glucose oxidase plus peroxidase and alkaline phosphatase.
  • U.S. Patent Nos. 3,654,090, 3,850,752, and 4,016,043 are referred to by way of example for their disclosure of alternate labeling material and methods.
  • the Smadl cDNA was cloned into the Sall/Pstl sites of pBGT9 vector to generate the pGBT9/Smadl bait plasmid.
  • human U2 OS osteoblast-like pACT cDNA library was screened following the procedure provided by the manufacture (Clontech, CA).
  • a full length mouse Hoxc-8 cDNA w as subcloned into the pACT vector at Xhol and EcoRl sites.
  • the pACT/Hoxc-8 was cotransformed with pBGT9/Smadl into Y 190 and the colonies were assayed for ⁇ -galactosidase expression using both colony lift filter assay and liquid assay.
  • GST glutathione S-transferase
  • GST-Smad2 and -Smad4 were digested with
  • the labeled Smadl protein was confirmed by SDS-PAGE.
  • Smadl -containing lysate (5 ⁇ l) was mixed with a n equivalent amount (1 ⁇ g) of GST alone or GST-Hoxc8.
  • Hoxc-8-containing lysate was mixed with GST alone or GST-Smadl .
  • the samples were incubated for 30 min on ice before GST-agarose diluted in NENT buffer (50 ⁇ l) was added to each sample and followed by a 30 min incubation at 4°C.
  • the Sepharose beads were washed four times in a PBS/0.1% TritonX- 100 solution, and bound proteins were eluted by incubation in 2X SDS-buffer for 5 min at 10°C.
  • the labeled Smadl protein in vitro translated lysate (1 ⁇ g) was loaded as input together with the eluted samples on a 12.5% SDS-PAGE.
  • HA-tagged Hoxc-8 was subcloned from pACT2/Hoxc-8 into a mammalian expression vector pcDNA3 (Invitrogen) a t
  • Expression vectors for FLAG-tagged Smadl and Smad4 were provided by Dr. Rik Derynck (University of California, San Francisco, CA).
  • Expression plasmids for constitutively active BMP type IA (ALK3) and IB (ALK6) receptors were provided by Dr. Jeffrey L. Wrana (Hospital for Sick Children, Canada).
  • COS-1 cells were transfected with expression constructs using Tfx-50 according to the manufacturers instructions (Promega). Cells were lysed 48 h post-transfection, and lysates were immunoprecipitated with anti-HA antiserum (Babco) and immunoblotted with anti-FLAG M2 (Eastman Kodak).
  • the osteopontin promoter from region -266 to - 1 was amplified by PCR from CH10T1/2 cell genomic DNA and cloned into S l and Xhol sites of the pGL3-basic vector (Promega) to generate a luciferase reporter construct (OPN-266).
  • Hox-pGL3 reporter bearing th e Hoxc-8 binding site (-290 to -166) was constructed using the s ame strategy but was put into the pGL3-control vector (Promega).
  • the Hox recognition core, TAAT was replaced with GCCG in Hox-pGL3 by PCR to create mutant Hox- ⁇ GL3 (mHox-pGL).
  • Smadl fused with a nuclear localization signal were constructed by PCR- based strategy into the cytomegalovirus (CMV) promoter-based mammalian expression vector, pCMV5.
  • CMV cytomegalovirus
  • pCMV5 cytomegalovirus
  • Each construct contained one of the following regions: Smadl-NL (amino acids 3 -276) , Smadl-L (aa 145-267), and Smadl-M (101-191 ).
  • Hoxc-8 w as subcloned from pACT2/Hoxc-8 into a mammalian expression vector, ⁇ cDNA3 (Invitrogen).
  • C3H10T1/2 cells (5x l 0 4 cells/well in 12-well culture dishes) were transfected with 0.5 mg of OPN266 luciferase reporter plasmid and different expression plasmids using Tfx-50 as described (Shi and Yang et al., 1999).
  • the Tet-Regulated Expression System (Gibco) was u sed to produce Smadl mutants expressing cell lines in 2T3 osteoblast precursor cells (Harris et al., 1996).
  • a NLS-linked Smadl -NL, Smadl-L, or Smadl-M was subcloned from pCMV5 into pTet- Splice vector (Gibco).
  • Two mg of pTet-splice/Smadl -NL, Smad l -L, Smadl-M, or pTet-splice (control), 2 mg of pTet-tTAk, and 40 ng of pcDNA3 (Clontech) were co-transfected and positive clones
  • RNA or mRNA from control and Smad l expressing cell lines was isolated with STAT-60 (Tel-Test) or with MicroPoly(A)Pure (Ambion) following manufacturers' instructions.
  • Northern blotting was performed using Rapid-Hyb buffer (Amersham) according to the manufacturer' s directions.
  • the osteopontin probe was PCR amplified with cDNA from C3H10T 1/2 cells as template.
  • the collagen type 1(a) and osf-2/cbfal probes were kindly provided by Dr. Harris (Univ. of Texas).
  • yeast two-hybrid system w as used to identify transcription factors that interact with Smadl in
  • Figure 1A illustrates the growth properties of the two hybrid system, demonstrating a specific interaction of Smadl with Hoxc-8 in vivo, which was further confirmed by ⁇ -gal liquid assay (Figure IB).
  • the yeast bearing both Smadl and Hoxc-8 plasmids grew on medium deficient in Trp, Leu, and His.
  • the full length Hoxc-8 fused with the Gal4 DNA binding domain was tested in the two hybrid system, it showed a much stronger interaction with Smadl ( Figure 1A and B).
  • gluathione S-transferase (GST) pulldown experiments were also performed with [ 35 S] methionine-labeled Hoxc-8 and a GST-Smadl fusion protein.
  • GST-Smadl fusion protein As shown in Figure 1C, Hoxc-8 was successfully co-precipitated with the purified GST- Smadl fusion protein, but not with the GST alone, demonstrating a direct interaction between the two proteins.
  • the present invention demonstrates the direct interaction between Smadl and Hoxc-8 and reveals the Smad l - mediated transcriptional mechanism in BMP-2/4-induced skeleton development. To examine the effect of the interaction on the
  • BMP-2 stimulates phosphorylation of Smadl , and phosphorylated Smadl in turn binds to Smad4 and takes th e complex into the nucleus. It is of interest whether Smadl, Smad4, or the complex of Smadl and Smad4 also interacts with Hoxc-8 in cells.
  • COS-1 cells were transiently co-transfected with expression plasmids for FLAG-Smadl, FLAG-Smad4, HA-Hoxc-8, and/or constitutively active BMP type IA receptor, ALK3 (Q233D). The cell lysates were immunoprecipitated with anti-HA antibody and immunoblotted with anti-FLAG antibody.
  • FIG. 2C demonstrates that Smadl (lane 3), Smad4 (lane 5) or both (lane 7) were co- immunoprecipitated with HA-Hoxc-8 in cells.
  • Co-transfection of ALK3 (Q233D) enhanced the interaction of Smadl (lane 4) or Smad4 (lane 6) with Hoxc-8.
  • ALK3 (Q233D) did not have any significant effect on Smadl (lane 3), Smad4 (lane 5) or both (lane 7) were co- immunoprecipitated with HA-Hoxc-8 in cells.
  • Co-transfection of ALK3 (Q233D) enhanced the interaction of Smadl (lane 4) or Smad4 (lane 6) with Hoxc-8.
  • ALK3 (Q233D) did not
  • EXAMPLE 12 Osteopontin promoter contains a Hoxc-8 binding element
  • the BMP-2 inducible genes w ere examined. Through a comparison of promoter sequences, putative Hox binding sites were found in four BMP-2 responsive bone matrix protein genes, bone sialoprotein, osteopontin, osteonectin and osteocalcin, which have served as marker genes for osteoblast differentiation. The osteopontin promoter was examined, since its mRNA expression was rapidly activated in response to BMP-2 treatment in C3H10T1/2 mesenchymal cells. There are five putative Hox binding sites, with a core sequence of Ta/tAT, within the first 382 bp of the 5' flanking region in osteopontin gene (Figure 3A). When the 212 bp DNA fragment from -382 to - 1 70
  • the specificity of the Hoxc-8 binding to the DNA was determined by a gel shift competition assay (Figure 3D). Unlabeled Hoxc-8 DNA binding element inhibited the shifted b and in a concentration dependent manner ( Figure 3E) in which a 1 00 - fold excess of the specific cold probe eliminated the Hoxc-8 binding, whereas a 100-fold excess of the Msx-2 DNA binding element did not.
  • Msx-2 is a homeodomain-containing protein, b u t it does not belong to the Hox family.
  • the Msx-2 DNA binding element was identified from the osteocalcin promoter, and its flanking regions of the core sequence is different from Hoxc-8 binding site.
  • Hoxc-8 binds to only one of the TAAT core sequences (-206 to -180), suggesting that the flanking regions are also important for Hoxc-8 binding.
  • Hoxc-8 binding site including its flanking regions, is highly conserved in chicken, mouse, pig and human.
  • the other four putative Hox sites may be involved in other homeodomain protein binding or may not be authentic Hox binding sites.
  • Hoxa-9 was chosen as a well characterized homeobox DNA binding protein to examine its interaction with different Smad proteins.
  • Msx-1 and Msx-2 were also used for gel shift assays for the same purpose.
  • Msx-1 and Msx-2 found at different loci than the Hox gene clusters, are involved in development of teeth. The expression of both genes is coordinately regulated by BMP-2 and BMP-4.
  • Hox binding site mediates BMP-induced transcription To examine whether Hoxc-8 binding site functions a s
  • a 266 bp osteopontin promoter fragment containing the Hoxc-8 binding site was cloned into th e pGL3-basic luciferase reporter vector to generate OPN-266 reporter plasmid.
  • Transfection of the OPN-266 construct in C3H10T1/2 mesenchymal cells showed that the reporter activity was stimulated moderately when Smadl or Smad4 expression plasmids were co-transfected.
  • the luciferase activity w as significantly enhanced when the OPN-266 reporter construct w as co-transfected with ALK3 (Q233D), Smadl, and Smad4 expression plasmids.
  • the ALK3 (Q233D)-induced transcriptional activity was completely abolished when Hoxc-8 w as overexpressed.
  • a shorter osteopontin promoter fragment containing the Hoxc-8 binding site was linked to a luciferase reporter vector under the control of the SV40 promoter (Hox- pGL3) or the tk minimal promoter (Hox-tk).
  • Hox-pGL3 the SV40 promoter
  • Hox-tk the tk minimal promoter
  • Co-transfection of the Hox-pGL3 construct into C3H10T1/2 mesenchymal cells with ALK3 (Q233D) or ALK6 (Q203D) luciferase reporter activity was induced more than 13- and 11-fold, respectively.
  • overexpression of Hoxc-8 suppressed the ALK3 (Q233D)-induced or ALK6 (Q203D)-induced reporter activity.
  • the wild type Smadl and its deletion mutants, as well as the empty bait (control, Figure 5A) were individually transformed into yeast cells containing a Hoxc-8 prey plasmid and ⁇ -gal activity was determined.
  • the wild type and mutants carrying MHl and/or the linker domains interacted with Hoxc-8, and showed a significantly increased ⁇ -gal activity compared with the
  • a homeodomain is responsible for the Hoxc-8 association with Smad l
  • Hox proteins have a similar homeodomain (HD) in common, consisting of a highly conserved DNA binding motif of 6 0 amino acids (Sharley, et al., 1995). Besides the homeotic domain that lies from amino acids (aa) 149 to 209, Hoxc-8 contains two other conserved peptide regions: an ocatpeptide (aa 1-8) and a hexapeptide (aa 137- 142) (Le Mouellic et al, 1988). The hexapeptide of Leu-Met-Phe-Pro-Trp-Met lies upstream from the homeodomain and is presumably involved in the interaction with Hox-assisting cofactors (Phelan et al, 1995 and Sharley et al., 1997). A recent study has revealed a direct contact between th e pentapeptide of Hoxb-1 and its DNA binding partner, the Pbx l protein (Piper, et al., 1999).
  • homeodomain may be directly involved in the Hoxc-8-Smad l interaction.
  • homeodomain alone ( Figure 6A, 149-209) is sufficient to support a strong interaction.
  • deletions encoding homeodomain ( 149-209) or homeodomain and its C-terminal flanking sequence (HDC, 1 5 1 - 242, Figure 6) were cloned into a bacterial expression vector to make mutants of the Hoxc-8 fusion proteins.
  • HD-containing deletion mutants of Hoxc-8 ( Figure 6B-D) were tested for their binding to DNA in the presence of either wildtype or mutant Smadl .
  • purified GST-HD and GST-HDC bound to the DNA probe (lanes 4 and 9, respectively).
  • Smadl-L and Smadl-M were also cloned into a tetracycline- regulated expression system. These plasmids and a control vector were permanently transfected into 2T3 cells, a well characterized osteoblast precursor cell line (Ghosh-Choudhury et al., 1996 and Chen, et al., 1998). Five to ten tetracycline-regulated positive clones were selected by slot blotting and Northern hybridization using corresponding cDNA probes. Figure 8B demonstrates that in three of the clones, expression of the Smadl fragments w as regulated by tetracycline.
  • Alkaline phosphatase activity is a hallmark in bone formation, and induction of its activity in progenitor cells m arks the entry of a cell into the osteoblastic lineage.
  • Stable expression of Smadl-NL, Smadl-L or Smadl-M by withdrawal of tetracycline effectively stimulates alkaline phosphatase activity in a time- dependent manner, whereas alkaline phosphatase activity remained unchanged in control cells permanently transfected with pTet-Splice vector (Figure 9C).
  • Figure 9D stable expression of those Smadl fragments in 2T3 cells induced bone mineralization
  • Hoxa-9 protein also binds to the osteopontin Hox binding site and Smadl inhibits its binding.
  • the protein domain of Hoxc-8 interacting with Smadl has been mapped using a yeast two-hybrid system and a gel shift assay.
  • the Hoxc-8 homeodomain a well conserved DNA binding motif, interacts with Smadl .
  • BMP-2/4 may either turn on or off gene transcription depending on the spatial and temporal expression of these Hox and homeodomain proteins as well as the promoter context ( Figure 4D), since Hox and homeodomain proteins function as both activators and repressors. This sophisticated regulation mechanism may explain why there has been no BMP-2/4 DNA response element characterized.
  • BMP-2/4 may stimulate mesenchymal cell differentiation by regulating binding of Hox o r homeodomain proteins from their DNA binding sites.
  • the pre sent invention demonstrates that BMPs induce the interaction between Smadl and Hoxc-8 protein, stimulating osteoblast differentiation in precursor cells.

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IIMURA T., ET AL.: "HOMEOBOX GENE EXPRESSION DURING BONE FORMATION INDUCED BY BMPA.", ANNALS OF THE NEW YORK ACADEMY OF SCIENCES, NEW YORK ACADEMY OF SCIENCES., US, vol. 785., 1 January 1996 (1996-01-01), US, pages 274 - 277., XP002921338, ISSN: 0077-8923 *
KANZLER B., ET AL.: "HOXA-2 RESTRICTS THE CHONDROGENIC DOMAIN AND INHIBITS BONE FORMATION DURING DEVELOPMENT OF THE BRANCHIAL AREA.", DEVELOPMENT, THE COMPANY OF BIOLOGISTS LTD., GB, vol. 125., no. 14., 23 January 1998 (1998-01-23), GB, pages 2587 - 2597., XP002921336, ISSN: 0950-1991 *
NISHIMURAT R., ET AL.: "SMAD5 AND DPC4 ARE KEY MOLECULES IN MEDIATING BMP-2-INDUCED OSTEOBLASTIC DIFFERENTIATION OF THE PLURIPOTENT MESENCHYMAL PRECURSOR CELL LINE C2C12.", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY, US, vol. 273., no. 04., 23 January 1998 (1998-01-23), US, pages 1872 - 1879., XP002921337, ISSN: 0021-9258, DOI: 10.1074/jbc.273.4.1872 *
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